Gonadotrophin stimulates phosphorylation of mitochondrial PHB in GCs. (A) GCs were treated with or without FSH (100 ng/mL) and testosterone (30 ng/mL) or in combination (FSH+testosterone) for 48 h. Parallel controls (C) of GCs were maintained without any treatments. After 48 h, 80 μg of protein purified from the respective mitochondrial fractions were focused in the first dimension on IPG 4–7 strips for 60 kV-h using a Bio-Rad Protean IEF Cell and second dimension followed by the Western blot analysis. PHB antibody was used to detect protein spots corresponding to PHB. (B) Alkaline phosphatase-treated GCs. GCs were cultured in the presence of FSH+testosterone for 48 h. Mitochondrial fractions were collected and treated with and without alkaline phosphatase followed by separation on 2D gel electrophoresis, and PHB spots were identified by Western blot analysis. (C) Representative Western blot analyses of mitochondrial protein levels PHB, pPHB (T258/259 and Y249), and OPA1 in GCs induced by FSH under PHB silencing experimental conditions. After various treatments, mitochondrial fractions were isolated, and pPHB and OPA1 were analyzed by Western blot analyses. Equal amounts of protein (25μg) were applied to each lane, and Western blot analyses were analyzed for PHB, pPHB, and OPA1. Porin was used as an internal control for mitochondria. (D) The bar graphs represent the relative percentage change in expression levels of PHB, pPHB, and OPA1 as a ratio of the protein levels normalized by porin from three independent experiments as mean±s.e.m. All are significantly different (P≤0.05) except with same alphabets (a, b, c, d, e, and f). (E) The bar graphs represent the relative percentage change in expression of mitochondrial PHB/porin and pERK/ERK in GCs induced by FSH under PHB silencing experimental conditions. After various treatments, mitochondrial protein was isolated and analyzed by Western blot analyses for PHB, pERK, and ERK. Porin was used as an internal control for mitochondria. The bar graphs represent the mean±s.e.m. of results from three independent experiments (n=3). All are significantly different (P≤0.05) except with same alphabets (a and b).
